UNIQUELY LABELED AMINO ACIDS FOR PROTEIN NMR

用于蛋白质 NMR 的独特标记氨基酸

• 批准号: 6271612
• 负责人: PAUL E YOUNG
• 金额: $ 18.63万
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271612
• 关键词: aminoacid; chemical structure function; chromatography; conformation; fibronectins; hormone analog; insulin; method development; methyl group; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein structure

The major objective of this proposal is to develop and apply methods to elucidate the dynamic structure of proteins in solution. It is new generally recognized that proteins do not have static structures and that X-ray data must be supplemented by motional information about side-chain and backbone atoms if a more complete understanding of the nature of protein interactions is to be achieved. We propose to synthesize a variety of 13C- and 2H-labeled amino acids and incorporate them into HIV-1 protease, despentapeptideinsulin amide, and the 9th and 10th type III domains of fibronectin. These molecules will then be studied by high field (200 600 MHz) nuclear magnetic resonance (Nmr) spectroscopy. We are especially interested in studying side-chain motion in these proteins and therefore our primary purpose is to synthesize amino acids labeled at every other side-chain carbon with 13C. Then with site- specific T1, T2, and long-range NOE data in hand, we hope to be able to paint a detailed picture of the structure and dynamics at and around these labeled sites. Recent work suggests that in many proteins flexibility may play a crucial role in determining enzymatic activity. Thus, for example, in proteins such as HIV-1 Protease, flexibility in the conformationally mobile "flap" region may control on-off access of the substrate and an understanding of this motion may provide information vital ta the rational design of protease inhibitors. To implement these projects, I am requesting support for two undergraduate students for four years. They will bear primary responsibility for the synthesis of all the labeled amino acids, peptides and insulin derivatives and they will routinely purify and analyze their products by chromatography and nmr. York's 200 MHz broadband FT-nmr, Sun Sparc5 workstation networked to an SGI with Biosym nmr processing/modeling software, and our solid phase peptide synthesizer will enable these students to participate fully in preparing, characterizing, and analyzing their 13C- and 2H- labeled products with the aid of forefront technology not generally available to undergraduates.

这项建议的主要目的是发展和应用方法